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CFB Practicals > Tuberculosis 1: Mycobacteria And Diagnosis Of Mycobacterial Infections > Flashcards

Tuberculosis 1: Mycobacteria And Diagnosis Of Mycobacterial Infections Flashcards

1
Q

Mycobacterium

A
  • ***Gram +ve Bacilli
  • ***Obligate aerobe
  • Slow growth rate
  • High lipid content of cell wall —> ↓ permeability to antibiotics + staining properties
  • Acid-fast staining —> Ziehl-Neelsen stain
  • Unique antibiotic susceptibility patterns
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2
Q

Classification of mycobacteria

A
  1. Mycobacterium tuberculosis complex
    - M. tuberculosis (commonest)
    - M. bovis
    - M. bovis BCG (bacillus Calmette-Guerin)
  2. Mycobacterium leprae
    - uncultivable with routine technique
    - diagnosis: Clinical, Histopathology, Nucleic acid amplification
  3. Other Mycobacteria (Non-tuberculous mycobacteria NTM / Mycobacteria other than tubercle bacillus MOTT / Atypical mycobacteria)
    - Runyon classification previously based on **growth rates + formation of **pigments —> Definitive identification: Molecular techniques
    - Runyon group 1: **Photochromogens —> produce pigments in light (e.g. M. kansaii)
    - Runyon group 2: **    Scotochromogens —> produce pigments in / without light (e.g. M. scrofulaceum)
    - Runyon group 3: **Non-chromogens —> do not produce pigments (e.g. M. avium complex)
    - Runyon group 4: **    Rapid growers —> visible growth within 7 days after subculture (e.g. M. fortuitum complex)
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3
Q

Diseases caused by mycobacteria

A
  • Very diverse
  • Opportunistic

M. tuberculosis:

  • ***Pulmonary + Extrapulmonary TB
  • Primary + Post-primary TB

M. leprae:
- ***Leprosy

NTM:

  • Pulmonary infections: M. kansasii
  • Lymphadenitis: M. scrofulaceum
  • ***Skin + soft tissue infections: M. marinum
  • Disseminated infection in immunocompromised: M. haemophilum
  • Catheter-related infections: Rapid growers
  • ***Nosocomial infection (e.g. contaminated devices, post-injection abscess, inadequate sterilisation of equipment): Rapid growers
  • Outbreaks associated with heater-cooler units in cardiac surgery: M. chimaera
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4
Q

Clinical specimen

A

M. tuberculosis: ***Always significant
Other mycobacteria: Significant if in sterile sites e.g. blood, tissue
NTM: maybe Colonisation if in sputum / superficial wound swabs

—> Need to correlate with **clinical + **radiological findings

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5
Q

Laboratory identification of Mycobacteria

A
  1. Conventional: Physiological + Biochemical tests
  2. MALDI-TOF MS: Rapid identification possible but accuracy depends on quality of MS database
  3. ***Molecular techniques (PCR + Sequencing): Most reliable
  4. Positive bacterial culture
    - **gold standard in microbiological diagnosis in most cases
    - **    very sensitive
    - allow exact species identification + antibiotic susceptibility testing
    —> antibiotic treatment very different for different species esp. NTM
    - allow antibiotic resistance detection esp. M. tb
    - allow epidemiological typing in outbreak investigations
    - ***however, slow growth rates + low bacterial load
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6
Q

***Drug resistance M. tuberculosis

A
  1. MDR-TB
    - Isoniazid + Rifampicin
  2. XDR-TB
    - MDR + any Fluoroquinolone + >=1 of 3 injectable second-line drugs (Amikacin, Kanamycin, Capreomycin)
  3. TDR-TB
    - All 1st + 2nd line drugs
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7
Q

Antimicrobial susceptibility testing

A
  1. M. tuberculosis
    - **Phenotypic method: culture isolate in presence of antimicrobial agents —> look for inhibition of growth
    - **    Genotypic assay: detect specific genes but resistance can be mediated by different gene mutations / multiple mechanisms which are not detected
  2. M. leprae
    - Routine testing not possible
  3. NTM
    - no standardised testing method
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8
Q

***Laboratory diagnostics of Mycobacterial infections

A
  1. ***Clinical suspicion
  2. ***Radiology
  3. ***Acid-fast stain
  4. ***Culture
  5. Tuberculostearic acid (TBSA) (not commonly used now)
  6. Adenosine deaminase (ADA)
  7. Urine lipoarabinomannan (LAM) antigen
  8. Antibody detection
  9. Tuberculin skin test
  10. In vitro interferon-γ release assay (IGRA)
  11. ***Nucleic acid amplification

Empirical treatment against TB may be given even infection not definitively confirmed by laboratory investigations, based on

  • clinical picture
  • suggestive epidemiological, radiological, pathological, other laboratory findings

NTM: Positive bacterial culture / PCR

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9
Q

Clinical specimens for diagnosis of mycobacterial infections

A

Quality + Quantity (e.g. volume of CSF)

Respiratory specimens:

  1. ***Sputum
  2. Lower respiratory tract: ***Bronchoalveolar lavage +/- Transbronchial biopsy / Transthoracic needle biopsy of lung
    - when lack of sputum / respiratory symptoms
    - failure of less invasive procedures
    - certain forms of TB e.g. Endobronchial TB
    - exclusion of concurrent infections / non-infectious pathologies e.g. malignancy
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10
Q

Diagnosis of extrapulmonary TB

A

AFB smear: ***Low sensitivity

  1. Tissue biopsy at infection sites
  2. Nucleic acid amplification tests (not necessarily high sensitivity)
  3. Histopathology
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11
Q

Clinical syndromes of TB

A
  1. Epidemiological context e.g. age, contact
  2. Radiological features
    - NOT distinguish TB from NTM
    - pulmonary TB not limited to lung apices
    - chest radiograph can be ***normal in endobronchial TB
  3. Underlying conditions
    - immunocompromised
    - HIV
    - treatment with biologics
  4. Other relevant lab findings
    - ***CSF cell counts + biochemistry in patients with meningitis
  5. Latent vs Active TB
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12
Q

***Latent vs Active TB

A

Latent TB (LTBI):

  • ***Negative bacteriological diagnosis
  • ***Suspicion from radiological findings
  • diagnosis: **Immunological tests e.g. **tuberculin skin test, ***IGRA
  • treatment: ***1 / 2 drug regimen to ↓ recurrence risk

Active TB:

  • ***Positive (more likely) bacteriological diagnosis
  • Immunological tests cannot differentiate active vs latent
  • treatment: ***standard 4 drug regimen
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13
Q

Microscopy / Smear

A
  • Simple, inexpensive, rapid
  • Operator-dependent
  • ***Low sensitivity: require >=10^4 organisms per ml of sputum, many patients have negative smear
  • cannot differentiate M. Tb from ***NTM by morphology alone
  • cannot differentiate living vs ***dead AFB using conventional acid-fast stain
  • ***quick guide to infectiousness of pulmonary TB: smear positive vs negative + infection control implications
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14
Q

Mycobacterial culture

A

Lager volumes of specimen better e.g. min 5ml of CSF

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15
Q

Serology

A

No use

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16
Q

Adenosine deaminase (ADA)

A
  • Derived from ***host lymphocytes + monocytes
  • ***not specific to M. Tb and its infection
  • used in pleural, pericardial, CSF as adjunct to standard bacteriological testing
17
Q

Urine lipoarabinomannan (LAM) antigen detection

A
  • Major ***glycolipid cell wall component of M. Tb
  • Inexpensive
  • done using ***ELISA / point-of-care testing using immunochromatographic format
  • high sensitivity in ***HIV patients with CD4 count <200/ml
  • poor sensitivity in non-HIV patients
18
Q

***Nucleic acid amplification tests e.g. PCR

A

Advantages:

  • Sensitivity: depends on bacterial load + type of specimens
  • Specificity 95-100%
  • ***Fast turnaround time
  • ***Species identification possible
  • Rapid detection of ***resistance genes
  • Fully automated in some commercial NAAT systems

Disadvantages:

  • Cost
  • ***Limited sensitivity for paucibacillary disease + extrapulmonary TB
  • Inhibitors in specimens may affect test
  • No definitive information on drug susceptibility / resistance
  • ***Only supplement but not replace conventional bacteriological testing
19
Q

Tuberculin skin test / Mantoux method

A

Procedure:

  • Intradermal injection of 0.1ml ***purified protein derivative (PPD)
  • RT-23 PPD: each 0.1ml consists of ***2 TU (tuberculin units)
  • wheal 6-10mm in diameter should be produced
  • inject another at once at a different site if 1st dose not administered correctly
  • record site, date, time of administration in medical record

Delayed-type hypersensitivity response:

  • person exposed to bacteria before will mount ***immune response in skin to bacterial proteins
  • read results at ***48-72 hours
  • record maximal transverse diameter (mm) of ***induration but not erythema
  • measured transversely to long axis of forearm at widest diameter
  • 0mm for absence of induration (X negative)
  • not equivalent to protective cell-mediated immunity against M. Tb

False negative:

  1. Technical (e.g. inadequate dose)
  2. Administration
  3. Reading (e.g. biased reader)
  4. Biological (e.g. viral infections, HIV, measles, disseminated TB, malignancy)

False positive:

  1. ***BCG vaccination
  2. ***NTM exposure

Cut-off value:

  • ***5mm: HIV-infected individual / immunocompromised patients
  • ***10mm: most individuals
20
Q

In vitro interferon-γ release assay (IGRA)

A

Alternative to TST

2 test formats:
1. QuantiFERON-TB Gold
2. T-SPOT TB
—> both uses peripheral blood for testing

Principle:

  • stimulate T cells of individual using mycobacterial antigens in vitro
  • release of **IFN-γ by T cells in response to **MTB-specific antigens
  • mononuclear cells from MTB-infected people will have higher level of IFN-γ production upon exposure to mycobacterial antigens

Antigens used:

  1. Early secretory antigenic target 6 (ESAT6)
  2. Culture filtrate protein 10 (CFP10)

Advantages:

  1. Both higher ***specificity than PPD as not being produced by BCG strain and most NTM
  2. Less affected by previous BCG vaccination / NTM infections

Disadvantages:

  1. Neither TST / IGRA distinguish ***latent vs active TB
  2. Also affected by underlying ***immune status e.g. HIV
21
Q

Histopathology + Cytology

A

Advantages:

  1. Useful when bacilli are few, yet still detectable host responses
  2. If very typical pathology seen (e.g. Langhans giant cells, caseous necrosis) —> suspect most likely causative agent

Disadvantages:

  1. Even if AFB present —> morphological findings does not definitely identify the exact species
  2. Host response can be modified by immune status (e.g. HIV: suppurative response rather than granulomatous inflammation)
  3. No information on drug resistance

CFB Practicals (24 decks)

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